Displacement chromatography of proteins using low molecular weight anionic displacers

ABSTRACT

A method for the purification of proteins by displacement chromatography on ion exchange media using low molecular weight displacers is disclosed. Several classes of low molecular weight anionic species are exemplified, including aromatic rings having sulfonic acid or carboxylic acid moieties attached thereon, sulfated sugar derivatives, anionic antibiotics, and dendrimeric polymers. Novel compounds useful as displacers are dendrimers of formula ##STR1## wherein R 1  is lower alkyl, n is 2 to 6 and similar dendritic polymers based thereon.

STATEMENT AS TO RIGHTS UNDER FEDERALLY SPONSORED RESEARCH

This invention was made with support from the National ScienceFoundation Grant No. BCS-9112481. The United States government may havecertain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of our earlier, applicationSer. No. 08/197,146 filed Feb. 16, 1994, now U.S. Pat. No. 5,478,924.

FIELD OF THE INVENTION

This invention relates to the displacement chromatography of proteinsusing low molecular weight anionic displacers and to a novel class ofdendritic polymer based polyelectrolytes useful for chromatography ofproteins.

BACKGROUND OF THE INVENTION

The displacement mode of chromatography was first recognized in 1906 byTswett, who noted that sample displacement occurred under conditions ofoverloaded elution chromatography. In 1943, Tiselius developed theclassifications of frontal chromatography, elution chromatography, anddisplacement chromatography. Since that time most developments andapplications, particularly those in analytical chromatography, havetaken place in the area of elution chromatography, and indeed the termchromatography without further qualification usually refers to elutionchromatography. Nonetheless, while the theory and practice of elutionchromatography has dominated the literature for the past fifty years,the theory and practice of displacement chromatography has occupied asmall niche in chromatographic science.

The two types of chromatography, elution and displacement, are readilydistinguished both in theory and in practice. In elution chromatography,a solution of the sample to be purified (in the case of the presentinvention, a protein) is applied to a stationary phase, commonly in acolumn. The mobile phase is chosen such that the sample is neitherirreversibly adsorbed nor totally unadsorbed, but rather bindsreversibly. As the mobile phase is flowed over the stationary phase, anequilibrium is established between the mobile phase and the stationaryphase whereby, depending upon the affinity for the stationary phase, thesample passes along the column at a speed which reflects its affinityrelative to other components that may occur in the original sample. Thedifferential migration process is outlined schematically FIG. 1, and atypical chromatogram is shown in FIG. 2. Of particular note in standardelution chromatography is the fact that the eluting solvent front, orzero column volume in isocratic elution, always precedes the sample offthe column.

A modification and extension of isocratic elution chromatography isfound in step gradient chromatography wherein a series of eluents ofvarying composition are passed over the stationary phase. In ionexchange chromatography, step changes in the mobile phase saltconcentration and/or pH are employed to elute or desorb the proteins.

Displacement chromatography is fundamentally different from desorptionchromatography (e.g., affinity chromatography, step gradientchromatography). The displacer, having an affinity higher than any ofthe feed components, competes effectively for the adsorption sites onthe stationary phase. An important distinction between displacement anddesorption is that the displacer front always remains behind theadjacent feed zones in the displacement train, while desorbents (e.g.,salt, organic modifiers) move through the feed zones. The implicationsof this distinction are quite significant in that displacementchromatography can potentially concentrate and purify components frommixtures having low separation factors while in the case of desorptionchromatography, relatively large separation factors are generallyrequired to give satisfactory resolution.

In displacement chromatography the eluent, (i.e. the displacer) has ahigher affinity for the stationary phase than does any of the componentsin the mixture to be separated. This is in contrast to elutionchromatography, where the eluent usually has a lower affinity. The keyoperational feature which distinguishes displacement chromatography fromelution or desorption chromatography is the use of a displacer molecule.In displacement chromatography, the column is first equilibrated with acarrier solvent under conditions in which the components to be separatedall have a relatively high affinity for the stationary phase. A largevolume of dilute feed mixture can be loaded onto the column andindividual components will adsorb to the stationary phase. That is, theydistribute from the feed solution onto the stationary phase, and remainthere. If all the components are to be resolved by displacement, thecarrier solvent emerges from the column containing no sample. The samplenow resides on the stationary phase and the position of each componenton the column is correlated with its relative affinity for thestationary phase. Conceptually, one can imagine each molecule of thecomponent with the highest affinity for the stationary phase displacinga molecule of a component having a lower affinity at a site on thestationary phase so that the individual components will ultimately bearranged on the column in sequence from highest to lowest affinity.

It will sometimes be advantageous to allow some of the components topass through the column with the carrier solvent; in this case only theretained feed components will be resolved by displacementchromatography.

Once the sample is loaded on the column, a displacer solution isintroduced. The displacer solution comprises a displacer in a suitablesolvent. The displacer is selected such that it has a higher affinityfor the stationary phase than does any of the feed components. Assumingthat the displacer and mobile phase are appropriately chosen, theproduct components exit the column as adjacent squarewave zones ofhighly concentrated pure material in the order of increasing affinity ofabsorption. This is shown schematically in FIG. 3. Following the zonesof purified components, the displacer emerges from the column. A typicalchromatogram from a displacement chromatography is shown in FIG. 4. Itis readily distinguished from the chromatogram of elution chromatographyshown in FIG. 2 by virtue of the fact that the displacer follows thesample and that the feed components exit the column as adjacent "squarewave" zones of highly concentrated pure material. Finally, after thebreakthrough of the displacer, the column is regenerated by desorbingthe displacer from the stationary phase to allow the next cycle ofoperation.

Displacement chromatography has some particularly advantageouscharacteristics for process scale chromatography of biologicalmacromolecules such as proteins. First, and probably most significantly,displacement chromatography can achieve product separation andconcentration in a single step. By comparison, isocratic elutionchromatography results in product dilution during separation. Second,since the displacement process operates in the nonlinear region of theequilibrium isotherm, high column loadings are possible. This allowsmuch better column utilization than elution chromatography. Third,column development per se requires less solvent than a comparableelution process. Fourth, displacement chromatography can concentrate andpurify components from mixtures having low separation factors, whilerelatively large separation factors are required for satisfactoryresolution in desorption chromatography.

With all of these advantages, one might presume that displacementchromatography would be widely utilized. However, displacementchromatography, as it is traditionally known, has a number of drawbacksvis-a-vis elution chromatography for the purification of proteins. Theterm "protein", as commonly understood in the art and as used herein,refers to polypeptides of 10 kDa molecular weight or more; according tothis convention, polypeptides of molecular weight below 10 kDa arecommonly referred to as oligopeptides. Two of the major problems are (1)regeneration of the column and (2) the presence of displacer in some ofthe purified fractions.

Since the displacement process uses a high affinity compound as thedisplacer, the time for regeneration and re-equilibration can be longcompared to elution chromatography. Furthermore, relatively largeamounts of solvent are often required during regeneration, effectivelyreducing any advantage over elution chromatography in solventconsumption.

The second problem, that of contamination by the displacer, has arisenbecause a common characteristic of displacers used in proteinseparations has been their relatively high molecular weight. Heretoforethe art has taught the use of high molecular weight polyelectrolytes todisplace proteins on the assumption that (as explained below) it isnecessary to have a large polyelectrolyte in order to ensure a higherbinding coefficient than the protein that is to be displaced. Highmolecular weight displacers exhibit both of the disadvantages enumeratedabove: they bind tightly to the stationary phase and therefore requireheroic conditions for regenerating the column, and traces of thedisplacer that may contaminate the product fraction are difficult toremove.

Therefore, it would be advantageous to have a class of displacers thatdid not require extensive regeneration of the column and that could bereadily removed from the product protein. There is one example in theart known to applicants of an attempt to use 2 kilodaltonpoly(vinylsulfonic acid) on polyethyleneimine-coated weak anion exchangeresin for the separation of conalbumin from ovalbumin. The experimentappears to have been successful in that the two proteins were separated[See Jen and Pinto Journal of Chromatography 519, 87-98 (1990)]. Howeverthe separation appeared to have been effected by a mixed mechanism ofelution and displacement chromatography, as discussed in a subsequentpaper [see Jen and Pinto Journal of Chromatographic Science 29, 478-484(1991)] in which the authors abandoned the poly(vinyl sulfate)displacers in favor of higher molecular weight dextran sulfate. In thissecond paper, Jen and Pinto demonstrate the superiority of the largerdextran sulfate over the smaller polyvinyl sulfate.

In a subsequent article, Jen and Pinto [Reactive Polymers 19, 145-161(1993), p.147] provide a table of all displacers used for thedisplacement chromatography of proteins on ion exchange stationaryphases. In their discussion of the results, they conclude, as before,that the 2 kDa polyvinyl sulfate partially displaces the second proteinand elutes the first.

It has now been surprisingly found that several classes of chargedspecies of very low molecular weight can function very efficiently asdisplacers for proteins in displacement chromatography.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a method for purifying aprotein, or several proteins, comprising loading the protein in asuitable mobile phase onto an ion exchange stationary phase anddisplacing the protein from the stationary phase by displacementchromatography using a displacer of molecular weight less than 2000.

In one embodiment the stationary phase is a cation exchange resin andthe displacer is a cationic species; in another embodiment thestationary phase is an anion exchange resin and the displacer is ananionic species of molecular weight less than 1620. In various preferredembodiments the anionic displacer includes compounds in which: a) one ormore sulfonic acid moieties are covalently attached to an aromatic ring(i.e. benzene disulfonic acid, toluene sulfonic acid, napthalenedisulfonic acid); b) one or more carboxylic acid moieties are covalentlyattached to an aromatic ring (phthalic acid); c) sulfated derivatives ofsugars (e.g. sucrose octasulfate); and d) dendrimers functionalized withsulfonates. In other preferred embodiments the displacer is apoly(quaternary ammonium) salt, or the displacer is an aminoacid ester,aminoacid amide, N-acylaminoacid, peptide ester, or N-acyl peptide,preferably a lower alkyl ester of lysine, lower alkyl ester of arginine,lower alkyl ester of N.sup.α -acylated lysine or lower alkyl ester ofN.sup.α -acylated arginine. Lower alkyl refers to linear, branched orcyclic, saturated hydrocarbon residues of six or fewer carbons. Thedisplacer may also be a cationic or anionic antibiotic, or a dendriticpolymer. When the displacer is a dendritic polymer, a preferreddisplacer is ##STR2## Sodium may be replaced with other common cationssuch as potassium, ammonium, or lithium.

Generally, the displacer may be advantageously selected fromelectrolytes whose characteristic charge (ν) and equilibrium constant(K) are such that when a coordinate system representing log K on theordinate and ν on the abscissa is created, a line constructed from apoint A on the ordinate axis through a point defined by the K and the νof the displacer has a greater slope than a corresponding lineconstructed from the same point A through a point defined by the K andthe ν of the protein to be purified. The point A corresponds in value tothe slope of the displacer operating line (Δ) in the system of interest.This will be explained in greater detail below.

In another aspect the invention relates to a method for purifying aprotein comprising loading the protein in a suitable loading solventonto an ion exchange stationary phase and displacing the protein fromthe stationary phase by displacement chromatography using a dendriticpolyelectrolyte displacer. The stationary phase can be a cation exchangeresin, in which case the polyelectrolyte will be a polycation, or thestationary phase can be an anion exchange resin, in which case thepolyelectrolyte will be a polyanion. Preferably, the polyelectrolyte isa poly(quaternary ammonium) salt. Another preferred dendritic polymer is##STR3## In another aspect, the invention relates to compounds offormula ##STR4## wherein R is --(CH₂)_(n) --N(R¹)₃ ⁺ X⁻, R¹ is loweralkyl n is 2 to 6, and X is as before. The compounds are useful asdisplacers in displacement chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a standard isocratic linearelution chromatography.

FIG. 2 is a typical chromatogram from elution chromatography.

FIG. 3 is a schematic representation of displacement chromatography.

FIG. 4 is a typical chromatogram from displacement chromatography.

FIG. 5 is a plot of equilibrium constant (K) versus characteristiccharge (v) for two proteins and two displacers of the invention.

FIGS. 6 through 11 are chromatograms of proteins using displacers of theinvention.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

A better understanding of the surprising discovery that small moleculescan be used effectively as displacers in the chromatography of proteinsis gained by briefly considering an improved mathematical model fordisplacement chromatography. Although this hypothetical construct isuseful to rationalize the phenomenon, it is not intended to limit thefull breadth of the invention.

The steric mass action (SMA) ion exchange model developed by one of theinventors, unlike other models, explicitly accounts for steric effectsin multicomponent protein equilibria and is able to predict complexbehavior in ion exchange displacement systems. A macromolecular solutelike a protein or a polyelectrolyte is presumed to have a multi-pointattachment on an ion-exchange surface and the number of interactionsbetween the absorbent surface and a single macromolecule is defined asthe characteristic charge of the solute molecule. The characteristiccharge of a solute is numerically equal to the number of saltcounter-ions displaced by the solute from the ion-exchange surface uponadsorption. However, in addition to the sites at which thepolyelectrolyte actually interacts, a large solute macromolecule boundto an ion-exchange surface also sterically hinders the adsorption ofmacromolecules of similar size onto sites underneath and adjoining thebound solute molecule. The number of sterically hindered saltcounter-ions on the surface (per adsorbed solute molecule), unavailablefor exchange with other solute molecules in the fluid phase is definedas the steric factor of the adsorbed macromolecule. Earlier treatmentsof mass action ion exchange equilibria assumed that the binding of amacromolecule to an adsorbent surface only affects a number of adsorbentsites equal to its characteristic charge. In fact, the steric shieldingof the stationary phase sites plays an important role in the non-linearadsorption behavior of macromolecules in ion-exchange systems.

The stoichiometric exchange of a solute molecule (protein orpolyelectrolyte) and the exchangeable salt counter-ions can berepresented by:

    C.sub.i +v.sub.i Q.sub.s Q.sub.i +v.sub.i C.sub.s          (1)

where C and Q are the mobile and stationary phase concentrations; v_(i)is the characteristic charge of the solute, and subscripts i and s referto the solute molecule and the salt counter-ion respectively. Theoverbar denotes bound salt counter-ions available for exchange with thesolute macromolecule in solution. The equilibrium constant, K_(i), forthe solute adsorbed on the ion-exchange surface is given by: ##EQU1##The equilibrium constant is a measure of the affinity of the molecule.The electro-neutrality condition on the stationary phase is given by thefollowing relation:

    .tbd.Q.sub.s +(v.sub.i +σ.sub.i)Q.sub.i              (3)

where σ_(i) is the steric factor of the displacer or protein.

Substituting equation 3 into equation 2 and rearranging yields thefollowing equilibrium relation for a single protein or displacer:##EQU2##

Thus, knowing the values of the mobile phase counter-ion concentrationC_(s), the column ion-bed capacity, Λ, and the model parameters for eachcomponent, one can easily generate a single component isotherm from theimplicit equation (4). The required model parameters for each componentare: characteristic charge, v_(i), steric factor, σ_(i) and equilibriumconstant K_(i). In order to employ this model for predictingdisplacement behavior, it is necessary to determine model parameters forthe proteins and the displacers.

Ion-bed capacity, Λ, can be measured in-situ using frontalchromatographic techniques [see Gadam et al., J. Chromatog. 630, 37-52(1993)].

For protein molecules exhibiting significant salt-sensitive retentionbehavior under low to moderate salt concentrations in the mobile phase,linear elution chromatography can be employed to determine two of thethree SMA model parameters (viz., characteristic charge and equilibriumconstant) using well established relationships for ion-exchange systems[see Kopaciewicz et al., J. Chromatog. 266, 3 (1983)]. Linear elutionexperiments are carried out at various mobile phase salt concentrationsin order to determine the characteristic charge (v_(i)) and equilibriumconstant (K_(i)) by the following equations:

    log k'=log (βK.sub.i .sup.v.sbsp.i)-v.sub.i log C.sub.s(5)

where, for a log k' v log C_(s) plot, slope=-v_(i) ; and intercept=log(βK_(i) ^(v).sbsp.i).

Having determined the characteristic charges and equilibrium constantsfor the proteins, the remaining SMA parameter, viz. steric factor,σ_(i), for the proteins is determined independently from a singlenon-linear frontal chromatographic experiment according to theexpression: ##EQU3## Many proteins exhibit significantly higher stericfactors relative to their characteristic charge, which is not surprisingin light of the conformational constraints in the protein molecules.Once the SMA parameters are obtained for a given protein, the model canthen be used to generate adsorption isotherms at any salt concentration.

While the determination of the characteristic charge and equilibriumconstant from linear elution data works well for moderately retainedproteins, it is quite difficult to characterize high affinity displacersin this fashion. Frontal chromatography, on the other hand, is wellsuited for parameter estimations for these high affinity compounds. Thecharacteristic charge of the displacer, v_(D), can be determined fromthe induced salt gradient using the following expression: ##EQU4##wherein n₁ is the total amount of ions displaced, n_(D) is the number ofmoles of displacer adsorbed on the stationary phase, C_(D) is the mobilephase concentration of polyelectrolyte displacer and ΔC_(s) is the stepincrease in the mobile phase counter-ion concentration upon displaceradsorption.

At sufficiently low mobile phase salt concentration the displacercompletely saturates the stationary phase material. Frontal experimentsunder these conditions can be employed to determine the steric factor,σ_(D), from the following expression ##EQU5## where Λ is the ion bedcapacity and Q_(D) ^(max) is the maximum stationary phase capacity ofthe polyelectrolyte displacer. Alternatively, the steric factor could bedetermined by measuring, for example, the sterically hindered sodiumions displaced by an ammonium front (analogous to bed-capacitymeasurement), n₂, as given by ##EQU6## The equilibrium constant for theion-exchange process is defined by equation 2. Once the characteristiccharge and steric factor are measured independently as described above,a frontal experiment is employed for the determination of theequilibrium constant K_(D). This experiment is performed under elevatedmobile phase salt conditions where the solute does not completelysaturate the bed. The equilibrium constant is directly calculated fromthe breakthrough volume using the independently determined values of thecharacteristic charge (v_(D)) and steric factor (σ_(D)) by theexpression ##EQU7## where β is the column phase ratio and C_(S) is theinitial salt concentration in the carrier. Once the characteristiccharge, steric factor and equilibrium constants are determined, theisotherms of the proteins and polyelectrolytes can be simulated usingthe SMA formalism described above.

According to the conventional wisdom based on results observed withderivatized polysaccharide displacers, a high molecular weight compoundwith a relatively high characteristic charge and a high steric factor tocharacteristic charge ratio is needed for protein displacementchromatography. There have been heretofore no clearly defined criteriafor selecting or determining the efficacy of one displacer over another.Using the mathematical model described above, it is now possible topredict the elution order of the feed components as a function of thecharacteristic charge and equilibrium constant of each of thecomponents, once the slope of the displacer operating line is known.

The mathematical criterion for effective displacement chromatography canbe reconstructed as a plot of log K_(i) vs. v_(i) (see FIG. 5). Theelution order in the isotachic displacement train can be thengraphically determined by constructing lines from the point on theordinate axis corresponding to the slope of the displacer operatingline, Δ, ##EQU8## through each of the points defined by the equilibriumparameters (characteristic charge and equilibrium constant) of thesolutes. The order of elution of the feed components corresponds to thecounter-clockwise order (i.e. increasing slopes) of these "affinity"lines. In equation (12) (C₁)_(d) is the concentration of salt that thedisplacer encounters (i.e. the carrier salt concentration), Q_(d) is theconcentration of the displacer on the stationary phase and C_(d) is theconcentration of displacer in the mobile phase.

Although applicants do not wish to be bound by this hypotheticalconstruct, it appears consistent with the discovery that small moleculescan be effective displacers, because size is not the critical parameter.According to the theory, any molecule whose K_(i) and ν_(i) places itcounterclockwise on the affinity plot from the protein in question willfunction as an effective displacer for that protein.

Consistent with this prediction, a number of low molecular weightdisplacers have been tested and found effective for proteindisplacement.

Dendritic polymers (also known as starburst polymers) arethree-dimensional, highly ordered oligomeric and polymeric compoundsformed by reiterative reaction sequences starting from smallermolecules--"initiator cores" such as ammonia or pentaerythritol. Withselected building blocks and propagation reactions, critical moleculardesign parameters such as size, shape, topology, flexibility and surfacechemistry can be precisely controlled at the molecular level. Thesyntheses proceed via discrete stages referred to as generations.Dendrimers possess three distinguishing architectural features: (1) aninitiator-core region, (2) interior zones containing cascading tiers ofbranch cells with radial connectivity to the initiator core, and (3) anexterior or surface region of terminal moieties attached to theoutermost generation.

The synthesis of a zero (12), first (14) and second [(16) shown inscheme 2] generation pentaerythritol based dendrimer was carried out asdescribed in detail later. The zero generation dendrimer is referred tofor convenience as PETMA4, PentaErythrityl (TriMethylAmmonium)₄, thefirst generation as PETMA12, and the second as PETMA36. ##STR5##

The SMA model equilibrium parameters for the zero, first and secondgeneration dendrimers were estimated in a 50×5 mm I.D. SCX column usingfrontal chromatographic techniques.

As can be seen from Table 1, approximately one-third of the total numberof charges on each of the dendrimers bind to the surface. The firstgeneration (PETMA12) and the second generation (PETMA36) dendrimersexhibited similar adsorption behavior, with similar values of σ_(D)/v_(D) and Q_(D) *v_(D) and marginal increases in Q_(D) with decrease insalt concentration.

                                      TABLE 1                                     __________________________________________________________________________    AVERAGE VALUES OF SMA PARAMETERS FOR                                          PENTAERYTHRITOL BASED DENDRIMERIC DISPLACERS                                        Salt (Na.sup.+)                                                                     Solute                                                                             Charac.                                                                            Steric                                                  Displacer                                                                           Conc. Concen.                                                                            Charge                                                                             Factor   Q.sub.D                                                                           Q.sub.D *v.sub.D                           (M.W.)                                                                              (mM)  C (mM)                                                                             (v.sub.D)                                                                          (σ.sub.D)                                                                   (σ.sub.D /v.sub.D)                                                           (mM)                                                                              (meq)                                      __________________________________________________________________________    PETMA4                                                                              20    15   1.5  2.6 1.73 140 210                                        (480)                                                                         PETMA4                                                                              50    21   1.6  1.5 0.94 183 293                                        (480)                                                                         PETMA4                                                                              50    4.18 N.D. N.D.                                                                              N.D. 177 N.D.                                       (480)                                                                         PETMA12                                                                             20    6.17 4.2  6.3 1.50 55.3                                                                              232                                        (1620)                                                                        PETMA12                                                                             75    6.17 4.2  N.D.                                                                              N.D. 51.5                                                                              216                                        (1620)                                                                        PETMA36                                                                             20    1.96 11   16.7                                                                              1.52 20.9                                                                              230                                        (5128)                                                                        PETMA36                                                                             50    1.96 10.5 N.D.                                                                              N.D. 18.3                                                                              192                                        (5128)                                                                        __________________________________________________________________________

As seen in Table 1, the second generation dendrimer PETMA36 has arelatively higher characteristic charge than the first generationdendrimer, but a similar σ_(i) /v_(i) ratio. According to theory,PETMA36 should act as an efficient displacer, and that was indeed foundto be the case. A two-protein displacement separation(α-chymotrypsinogen A and cytochrome C) using PETMA36 was carried out ina 100×5 mm cation exchange column. There was a reasonably good match oftheory and experiment. The experiment was repeated using purified(diafiltered) first generation pentaerythritol PETMA12 as the displacer.These displacements indicate that decreasing the molecular weight andnumber of charged groups on the dendrimers appears to have little effecton their efficacy as displacers. Extending the prediction one levelfurther, one would predict a zero generation dendrimer should also actas a protein displacer. This prediction runs counter to the conventionalwisdom of using high-molecular weight polyelectrolytes with highcharacteristic charges as displacers of proteins in ion-exchangesystems. (The zero generation dendrimer has a net charge of 4, acharacteristic charge of 1.7 and a molecular weight of 480 Da.)

The results of the displacement chromatography of the two-proteinmixture of α-chymotrypsinogen A and cytochrome-C with the zerogeneration dendritic displacer are shown in FIG. 6. As seen in thefigure, an excellent displacement separation of the two proteins isobserved in highly concentrated adjacent zones with sharp boundaries andrelatively minimal mixing. This result is truly revolutionary, and is ofprofound significance for implementation of displacement chromatographyfor large-scale protein separations.

It is seen that the zero, first and second generation dendriticpolyelectrolytes function as efficient displacers of proteins inion-exchange systems. More significantly the ability of a low molecularweight compound such as the `zero` generation dendrimer (M.W. 480) todisplace relatively high molecular weight proteins is quite exciting inthe current context of understanding displacement phenomena. Since themolecular weight and number of charged groups on the dendrimers appearto have little effect on their efficacy as displacers, it may be moreadvantageous to use a `zero` generation dendrimer as a displacer. Thesynthesis of these molecules is much easier and involves fewer steps;(hence they are cheaper). They have the additional advantage of easyseparation from any feed component zones during post-displacement,size-based downstream processing.

Low molecular weight anionic electrolytes also function effectively asdisplacers for proteins in anion exchange systems. Some examples of lowmolecular weight compounds which have been shown to be effectivedisplacers of proteins in anion exchange systems include (a) compoundsin which one or more sulfonic acid moieties are covalently attached toan aromatic ring (i.e. benzene disulfonic acid, toluene sulfonic acid,napthalene disulfonic acid); (b) compounds in which one or morecarboxylic acid moieties are covalently attached to an aromatic ring(phthalic acid); (c) sulfated derivatives of sugars (e.g. sucroseoctasulfate); and (d) dendrimers functionalized with sulfonates.

An example of a displacement chromatogram of β-lactoglobulins A and B onan 8 μm strong anion exchange column using 32.0 mM naphthalenedisulfonic acid (NDS) as a displacer in a 50 mM salt solution at pH 7.5is shown in FIG. 9. As seen in the figure, this low molecular weightanionic displacer (280 daltons) produced an efficient displacementseparation of the two proteins. The separation was characterized bysharp displacement boundaries between the protein zones as well as theprotein-displacer interface.

In contrast to high molecular weight displacers, the dynamic affinity oflow molecular weight displacers is more sensitive to operatingconditions. By employing an appropriate displacer concentration, one canprovide conditions whereby the weakly retained impurities are eluted inthe induced salt gradient, the target protein is displaced, and thestrongly retained impurities are desorbed in the displacer zone. Thistype of displacement is termed as "selective displacementchromatography". When a displacement experiment was carried out on an 8μm strong anion exchange column using 65 mM p-toluene sulfonic acid as adisplacer in a 50 mM salt solution at pH 7.5, both the proteinsβ-lactoglobulin A and β-lactoglobulin B were well-displaced by thedisplacer. However, when the displacer concentration was reduced to 46mM p-toluene sulfonic acid, the experiment resulted in the displacementof β-lactoglobulin B (target protein) and the desorption ofβ-lactoglobulin A (strongly retained impurity) in the displacer zone.Thus, by reducing the displacer concentration from 65 to 46 mM, theseparation was transformed from a traditional displacement separation toa selective displacement separation. The ability of low molecular weightdisplacers to act as selective displacers may have important advantagesfor bioprocessing of proteins.

Compounds containing carboxylic acid moieties can also be employed asanionic displacers. An example of a displacement separation ofβ-lactoglobulin A and B using 35 mM phthalic acid in a 50 mM saltsolution at pH 7.5 is shown in FIG. 10. Under these conditions,displacement with phthalic acid resulted in selective displacement ofβ-lactoglobulin B and desorption of β-lactoglobulin A in the displacerzone.

In addition to sulfonic and carboxylic acid containing compounds,sulfated sugar derivatives can be employed as efficient and non-toxicdisplacers. An example of a displacement separation of β-lactoglobulin Aand B employing 10 mM of sucrose octasulfate in a 50 mM salt solution,pH 7.5 is shown in FIG. 11. As seen in the figure, the mixture was verywell resolved into two components with minimal tailing ofβ-lactoglobulin A into the displacer zone.

Other low molecular weight electrolytes also appear to functioneffectively as displacers for proteins. For example, modified aminoacids and charge-bearing antibiotics can be used as displacers. Bymodified it is meant that the amino acid is altered so as to change itfrom amphoteric to either cationic (for cation exchange displacementchromatography) or anionic (for anion exchange chromatography). This ismost conveniently accomplished by esterifying the carboxylate to producecationic species or acylating the amine to produce anionic species.

Displacers whose charge is derived from a single carboxylate tend to beless effective anionic displacers because of their lower characteristiccharge at a pH commonly used in chromatography; as a result, they wouldhave to have an extremely high equilibrium constant to fallcounterclockwise from most proteins of interest on the affinity plot.For this reason, among amino acids, acylated taurine derivatives aremore likely candidates for anionic displacers.

Carboxyl-derivatized amino acids provide very effective cationicdisplacers. For example, carbobenzoxylysine methyl ester,benzoylarginine ethyl ester (BAEE) and arginine methyl ester are alleffective displacers in the displacement chromatography ofα-chymotrypsinogen and cytochrome-C. The first two have a single,positive charge; arginine methyl ester has two positive charges, and asa result, a higher affinity for the stationary phase. The resolution ofα-chymotrypsinogen and cytochrome-C in isotachic displacement iscomparable to the resolution obtained using high molecular weightdisplacers such as DEAE dextran. An example of a displacementchromatogram of α-chymotrypsinogen A and cytochrome C on an 8 micronstrong cation exchange column using 45 mM benzoyl arginine ethyl esterin a 50 mM salt solution at pH 6.0 is shown in FIG. 7. The modifiedamino acid displacers can be purchased in a very pure form at a costwhich is a small fraction of the cost of high molecular weightdisplacers. In addition, their small size provides them with bettertransport properties and faster kinetics.

Many antibiotics have the virtue that they are small enough to beremoved easily if found in desired protein fractions, but in addition,they can often be advantageously left in the protein fraction. In orderto achieve the desired combination of high characteristic charge andequilibrium constant, it appears that antibiotics having one or morestrongly dissociating functionalities are particularly useful. Suchantibiotics include the streptomycins, which have two guanidinefunctionalities. An example of a displacement chromatogram ofα-chymotrypsinogen A and cytochrome C on an 8 micron strong cationexchange column using 45 mM streptomycin sulfate (m.w. 581) in a 30 mMsalt solution at pH 6.0 is shown in FIG. 8.

The demonstration that aminoacid esters, dissociated antibiotics andzero generation dendrimers, all having molecular weights under 600, arehighly effective displacers confirms that molecular weights above 2000are not necessary for displacers for protein chromatography. Indeed wehave found no instance of a charged species of molecular weight below2000 that did not work, as long as the characteristic charge andequilibrium constant were such that the SMA analysis (shown in FIG. 5and explained above) predicted efficacy.

A displacement separation of a two-component protein mixture was alsocarried out using crude PETMA (12) as the displacer in a 100×5 mmcation-exchange column. Although the protein components were displacedand well separated in adjacent zones, the effluent profile exhibitedsimilar characteristics to earlier displacements with impureDEAE-dextran displacers. Most strikingly, the cytochrome-C zone wasconsiderably less concentrated in relation to the α-chymotrypsinogen Azone. Apparently, impurities in the displacer contributed to thedesorption of the proteins and depression of their isotherms. It istherefore believed advantageous to purify the dendrimers.

One characteristic of dendrimers is that a variety of terminal moietiesmay reside on the surface of the dendrimer. The terminal groups can bereadily converted to functionalities that provide the potential fordifferent utilities, and dendrimers possess a very high density of theseterminal moieties which reside in the final exterior layer. When theorganic groups on the surface of these compact molecules arefunctionalized to be charged groups, such as quaternary ammonium saltsand sulfonates, they exhibit higher affinity toward chromatographicmedia than some proteins, making them useful as a new type of displacerin chromatographic separation. The synthesis of the backbone of adendritic polyether is shown in Scheme 1 and its functionalizationpathway to novel poly(ether-amine)s is illustrated in Scheme 2. Thefunctionalization of dendrimeric precursors to provide anionicdendrimers (sulfonates) is shown in Scheme 3. The strategy used inScheme 1 is a modified procedure derived from the work of Hall andPadias [J. Org. Chem. 52, 5305 (1987)]. Pentaerythritol (PE) is also theinitiator core but1-methyl-4-(hydroxymethyl)-2,6,7-trioxabicyclo-[2.2.2]-octane (MHTBO) isused as the building block instead of the hydroxymethyl bicyclicorthoformate (HTBO). N,N-dimethylethanolamine is used as the synthon tointroduce tertiary amine sites. ##STR6##

Preparation of Dendrimeric Electrolytes

Hexane, tetrahydrofuran (THF), and 2-methoxyethyl ether (diglyme) weredried over sodium and distilled right before use. N,N-dimethylformamide(DMF) was purchased from Aldrich Chemical Co. in HPLC grade. All othersolvents and reagents were used without additional purification unlessspecified in the procedure.

Pentaerythrityl Tetrabromide, PE-Br(4) (compound 1)

In a 1 L, three-necked, round-bottom flask equipped with a mechanicalstirrer and a thermometer were placed pentaerythritol (26.0 g. 0.19 mol)and 200 mL of pyridine. Stirring was initiated and to the suspension,cooled in an ice-bath, was added p-toluenesulfonyl chloride (152.52 g,0.8 mol) as a solid at such a rate that the temperature did not riseabove 30° C. After the addition was completed the resulting slurry wasstirred at 35°-40° C. for another two hours. The slurry was then addedslowly to a vigorously stirred solution of 200 mL of water, 400 mL ofmethanol and 160 mL of concentrated hydrochloric acid. The crude whitepentaerythrityl toluenesulfonate was further cooled by adding more ice,filtered with suction and washed with 1 L of water and 200 mL of coldmethanol in two portions.

In a 1 L, three-necked, round-bottom flask fitted with a mechanicalstirrer, a thermometer and a condenser were mixed the slightly wetpentaerythrityl toluenesulfonate (about 140 g), sodium bromide (120 g,1.16 mol) and 300 mL of diethylene glycol. The mixture was then heatedto 140°-150° C. with slow stirring and reacted in this temperature rangeovernight. After being cooled to about room temperature, the mixture waspoured into 400 mL of water with stirring,-the precipitate was filteredwith suction and washed with 500 mL of water. The crude product wasdried under vacuum (1 torr) at 50° C. overnight and recrystallized fromacetone. Yield: 52 g (70%). Mp: 157°-160° C.

1-Methyl-4-(hydroxymethyl)-2,6,7-trioxabicyclo[2.2.2]octane (MHTBO,compound 2)

Pentaerythritol (13.6 g, 0.1 mol), triethyl orthoacetate (16.22 g, 0.1mol, 18.3 mL), pyridine p-toluenesulfonate (PPTS) (0.5 g, 2 mmol) and100 mL of dioctyl phthalate were mixed in a 250 mL, round-bottom flaskfitted with a regular distillation apparatus. The mixture was heated to130°-140° C. and ethanol was slowly distilled. When the amount ofethanol was close to the theoretical value the pressure was reduced to<0.1 torr and the product was distilled in vacuo. The white productwhich distilled crystallized in the condenser to give 13.6-14.9 g ofMHTBO. Yield: 85-93%. The compound can be recrystallized from toluenebut can be used directly. Mp: 110°-112° C.

PE-MBO(4) (compound 3)

In a 500 mL, three-necked flask equipped with a mechanical stirrer, athermometer and an addition funnel, under an argon atmosphere, potassiumhydride (2.8 g, 0.07 mol, 8.0 g of 35% suspension) was washed twice withhexane, the washings were decanted, and 100 mL of diglyme was added.After the mixture was cooled to 0° C. with stirring, a solution of 10.25g (0.064 mol) of MHTBO in 100 mL of diglyme was added dropwise and themixture was stirred at room temperature for three hours. Then a solutionof 5.82 g (0.015 mol) of pentaerythrityl tetrabromide in 100 mL ofdiglyme was added dropwise also at room temperature. The mixture washeated to reflux for 24 hours. The mixture was then poured into 600 mLof ice water and the precipitate was filtered, washed with water, anddried under vacuum (1 torr) at 50° C. overnight. A white solid (8.2 g,78%) was obtained. The Beilstein test for bromide was negative. Theproduct was finally recrystallized from 4:1 ethyl acetate/hexane. Yield:5.86 g, 55%. Mp: 220° C. slight shrinkage, 230°-244° C. melting.

PE-OH(12) (compound 4)

In a 250 mL, round-bottom flask PE-MBO(4) (6.0 g, 8.53 mmol) was mixedwith 100 mL of methanol and 1 mL of concentrated HCl. The mixture washeated slowly to reflux and kept under reflux for one hour. Methanol andmethyl acetate were distilled off until only about 1/3 of the solventremained. The white product was filtered and dried. Yield: 4.86 g (8.0mmol), 94%. Mp: 180° C. with shrinkage, 220°-235° C. melting.

PE-Tos(12) (compound 5)

In a 500 mL Erlenmeyer flask PE-OH(12) (2.24 g 3.68 mmol) was dissolvedin 40 mL of pyridine and cooled to 0° C. A solution of 21.2 g ofp-toluenesulfonyl chloride (0.11 mol, 30 equiv) in 100 mL of pyridinewas added dropwise through an addition funnel. The solution was stirredfor another hour at 0° C. and then left at room temperature for fourdays. The mixture was poured into 500 mL of ice water and the solventwas decanted after the precipitate agglomerated at the bottom of thebeaker. The crude product, 9 g, was dried under vacuum (1 torr) at 50°C. overnight and was then recrystallized from 4:1 ethanol/chloroform.Yield: 8.16 g (3.32 mmol), 90%. Mp: 130°-133° C.

PE-Br(12) (compound 6)

PE-Tos (12) (8.0 g, 3.25 mmol) was dissolved in 50 mL ofN,N-dimethylacetamide (DMAc) and 10.06 g of NaBr (98 mmol, 30 equiv) wasthen added. The resulting suspension was stirred and heated to 150° C.and kept at this temperature for another hour. The mixture was cooled toroom temperature and poured into ice water. The precipitate wasfiltered, dried under vacuum (1 torr) overnight and recrystallized fromethyl acetate. Yield: 3.40 g (77%). Mp: 150° C. with shrinkage,172°-177° C. melting.

PE-MBO(12) (compound 7)

In a 500 mL, three-necked, round-bottom flask fitted with a mechanicalstirrer, a thermometer and an addition funnel under an argon atmospherepotassium hydride (1.6 g, 40 mmol, 4.57 g of 35% suspension) was washedwith hexane twice, the washes were decanted and 100 mL of DMF was added.The mixture was cooled to 0° C. and a solution of 5.76 g of MHTBO(compound 2) (36 mmol, 24 equiv) in 50 mL of DMF was added dropwise. Theresulting suspension was stirred for three hours at room temperature.The dodecabromide (2.05 g, 1.5 mmol) was dissolved in 100 mL of DMF at50° C. and then was added rapidly dropwise to the reaction flaskimmediately while still warm. The mixture was heated to reflux for 24hours and then poured into ice water containing about 50 g of sodiumchloride. The precipitate was filtered and dried under vacuum (1 torr)at 40° C., overnight. The crude yield was quantitative (3.45 g) and theproduct was purified by column chromatography with silica gel as thestationary phase and a mixture of 1:1 ethyl acetate/hexane as theeluent. R_(f) value: 0.42. Yield: 2.9 g (1.25 mmol), 82%. The Beilsteintest for halogen was negative. Mp: 78° C. with shrinkage, 88°-90° C.gelation, 170° C. softening.

PE-OH(36) (compound 8)

In a 250 mL, round bottom flask were placed PEMBO(12) (4.4 g, 1.9 mmol),100 mL of methanol and 1 mL of concentrated HCl. The mixture was heatedto reflux for one hour. Methanol and methyl acetate were distilled untilonly 15-20 mL of the solution remained and the solution was transferredto a beaker. After the rest of the solvent was evaporated completely,the syrup was dried under vacuum (1 torr) to yield a foam. Yield: 3.35 g(1.66 mmol), 88%. Mp: 75° C. with shrinkage, 83°-85° C. gelation,220°-230° C. softening.

PE-TOS (36) (compound 9)

In a 500 mL Erlenmeyer flask PE-OH(36) (4.17 g, 2.05 mmol) was dissolvedin 180 mL of pyridine and cooled to 0° C. A solution ofp-toluenesulfonyl chloride (29.4 g, 0.15 mol, 75 equiv) in 200 mL ofpyridine was added dropwise. The mixture was stirred for another hour at0° C. and then left at room temperature for 7 days. The brown solutionwas poured into 2 L of ice water and the precipitate was filtered anddried under vacuum (1 torr) at 40° C. overnight. Yield: 14.36 g (1.88mol), 92%. Mp: 120° C. with shrinkage, 220°-245° C. melting.

PE-Br(36) (compound 10)

In a 250 mL, three-necked, round bottom flask were mixed 7.58 g (1.0mmol) of PE-Tos(36), 8.32 g (80 mmol, 80 equiv) of NaBr and 100 mL ofDMAc. The mixture was stirred and heated to 150° C. and kept at thistemperature for one hour. Then the mixture was cooled to roomtemperature and poured into 2 L of ice water with stirring. Theprecipitate was filtered, washed with another 500 mL of water and driedunder vacuum (1 torr) at room temperature overnight. Yield: 4.18 g, 98%.Mp: 48° C. with shrinkage. 52°-68° C. melting.

Tetrakis[((N,N dimethylamino)ethoxy)methyl]methane, PE-DMA(4) (compound11)

In a 500 mL, three necked, round-bottom flask equipped with a mechanicalstirrer, an addition funnel and a thermometer, potassium hydride (5.2 g,0.13 mol, 14.8 g of a 35% suspension) was washed with hexane twice underan argon atmosphere and 100 mL of DMF was added. When the mixture wascooled to 0° C. a solution of 10.7 g (0.12 mol, 6 equiv) ofN,N-dimethylethanolamine in 100 mL of DMF was added dropwise and stirredat room temperature for three hours. A solution of 7.8 g (0.02 mol) ofpentaerythrityl tetrabromide in 100 mL of DMF was added dropwise. Themixture was heated to 80° C. and reacted at 80°-90° C. for 12 hours.Then the temperature was raised to reflux for another 12 hours. Theresulting mixture was cooled to below 50° C. and poured into 300 mL ofice water. All of the solvents were removed on a rotavapor and theresidue was extracted with 800 mL of ethyl ether in a few portions, andthe combined extracts were dried over MgSO₄. After the ether wasevaporated, the crude product was distilled under vacuum (<0.1 torr) toobtain 5.7 g of oily liquid PE-DMA(4). The compound was further purifiedto a clear liquid on an Al₂ O₃ column using a mixture of 4:1 ethylacetate/hexane as the eluent. R_(f) value: 0.57. Yield: 4.66 g 59%. Bp:140°-143° C. (0.03 mmHg).

Tetrakis[((N,N,N-trimethylammonium iodide)ethoxy)methyl]methane, PE-TMAiodide (4) (compound 12)

In a 100 mL, three necked flask were placed PE-DMA(4) (2.3 g, 5.5 mmol)and 30 mL of THF under an argon atmosphere. The solution was cooled to0° C. and a solution of CH₃ I (9.3 g, 66 mmol, 12 equiv, 4.1 mL) in 20mL of THF was added dropwise. After the addition was completed themixture was stirred at room temperature for five more hours. Theyellowish precipitate was filtered, dried under vacuum (1 torr) at 60°C. overnight and finally recrystallized from methanol. The compound ishighly hygroscopic. Yield: 3.84 g, 70%.

PE-DMA(12) (compound 13)

The procedure is similar to the one used for PE-DMA(4). Potassiumhydride (2.8 g, 0.07 mol, 8.0 g of a 35% suspension) was washed withhexane twice under an argon atmosphere and 100 mL of DMF was added. Asolution of 6 mL (0.06 mol, 5.34 g) of N,N-dimethylethanolamine in 50 mLof DMF was added dropwise at 0° C. and the mixture was stirred at roomtemperature for three hours. PE-Br(12) (2.72 g, 32 mmol) was dissolvedin 150 mL of DMF at 50° C. and the solution was added while still warmthrough an addition funnel, dropwise. The mixture was heated to refluxfor 24 hours and poured into 300 mL of ice water. After all the DMF andwater were evaporated, the residue was extracted with 800 mL of ether inseveral portions and dried over MgSO₄. The ether solution was filteredand concentrated to about 200 mL. In a 300 mL, three necked flask, HClgas was introduced to the solution and the solvent was decanted when nomore salt was formed. The salt was washed with anhydrous ether twice,dried under argon for half an hour, dissolved in water and basified topH>10. The resulting aqueous solution was dried on a rotavapor and theresidue was extracted with 500 mL of ether and dried over MgSO₄. Afterthe ether was evaporated, 2.1 g of fairly pure product was obtained. Thecompound is a viscous oil; bp 220° C. at 0.02 torr. Yield: 72%.

PE-TMA iodide (12) (compound 14)

The procedure is identical with the one used for PE-TMA (iodide(4).PE-DMA (12) (2.04 g 1.4 mmol) was dissolved in 60 mL of THF. At 0° C. asolution of 3.2 mL (7.12 g, 50 mmol, 36 equiv.) of CH₃ I in 20 mL THFwas added dropwise. The mixture was stirred at room temperature foranother three hours. The yellow precipitate was filtered and dried undervacuum (1 torr) at 60° C. overnight. This salt cannot be recrystallizedfrom methanol and was further purified by ultrafiltration before beingtested as a displacer. Yield: 3.74 g, 84%.

PE-DMA(36) (compound 15)

This procedure is identical with the one used for PE-DMA(12). Potassiumhydride (2.8 g, 0.07 mmol, 8.0 g of 35% suspension) was washed twicewith hexane and 100 mL of DMF was added. At 0° C. a solution of 6.4 mL(5.7 g, 64 mmol, 80 equiv.) of N,N-dimethylethanolamine in 50 mL of DMFwas added and the mixture was stirred at room temperature for threehours. PE-Br(36) (3.43 g, 0.8 mmol) was dissolved in 100 mL of DMF andadded dropwise at room temperature. The mixture was then heated toreflux for 24 hours and poured into 200 mL of ice water. After allsolvents were removed, the residue was extracted with 800 mL of ether.The polyamine was converted to a salt by bubbling HCl gas into an ethersolution and then was freed by basifying the aqueous solution to pH>10.This compound is a very viscous syrup and is highly hygroscopic. Yield:1.86 g, 51%.

PE-TMA iodide (36) (compound 16)

The procedure is also identical to the one used for PE-TMA iodide(4) andPE-TMA(12). PE-DMA(36) (1.79 g, 0.39 mmol) was dissolved in 100 mL ofTHF and cooled to 0° C. A solution of 4.2 g (1.82 mL, 30 mmol, 75equiv.) of CH₃ I in 20 mL of THF was added and the mixture was stirredat room temperature for another three hours. The precipitate was thenfiltered and dried under vacuum (1 torr) at 60° C. overnight. Yield: 2.8g, 75%.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that other changes in form and details may bemade therein without departing from the spirit and scope of theinvention.

We claim:
 1. A method for purifying a protein comprising loading saidprotein in a loading solvent onto an ion exchange stationary phase anddisplacing said protein from said stationary phase by displacementchromatography using an anionic displacer of molecular weight less than1620.
 2. A method according to claim 1 wherein said stationary phase isan anion exchange resin and said anionic displacer is an anionicspecies.
 3. A method according to claim 1 wherein said anionic displaceris a compound containing an anion selected from the group consisting ofsulfate, sulfonate, acetate, and phosphate.
 4. A method according toclaim 1 wherein said anionic displacer is an anionic compound containingat least one sulfonic acid moiety.
 5. A method according to claim 4wherein each of said at least one sulfonic acid moiety is covalentlyattached to an aromatic ring.
 6. A method according to claim 5 whereinsaid anionic displacer is naphthalene disulfonic acid.
 7. A methodaccording to claim 5 wherein said anionic displacer is toluene sulfonicacid.
 8. A method according to claim 1 wherein said anionic displacer isan anionic compound containing at least one carboxylic acid moiety.
 9. Amethod according to claim 8 wherein each of said at least one carboxylicacid moiety is covalently attached to an aromatic ring.
 10. A methodaccording to claim 9 wherein said anionic displacer is phthalic acid.11. A method according to claim 1 wherein said displacer is an anionicderivative of a sugar.
 12. A method according to claim 11 wherein saidanionic displacer is a sulfated derivative of a sugar.
 13. A methodaccording to claim 11 wherein said sugar is sucrose.
 14. A methodaccording to claim 13 wherein said displacer is sucrose octasulfate. 15.A method according to claim 1 wherein said anionic displacer is ananionic antibiotic.
 16. A method according to claim 1 wherein saiddisplacer is a dendritic polyanion.
 17. A method according to claim 16wherein said dendritic polyanion is a sulfonate.